Precursor Ion Scan Mode Research Articles

High Specificity and Sensitivity Method for Quantitation of Anthocyanins by Precursor Ion Scan Using Liquid Chromatography‐Tandem Mass Spectrometry Technique

ABSTRACTAnthocyanins, primarily found in flowers, vegetables, and fruits, play a critical role in human and plant health. These polyphenolic pigments are predominantly found in plants in the form of glucosides linked to one or more sugar units and exhibit a broad range of structural diversity. This creates a complex spectrum of anthocyanins making it challenging to perform its absolute quantification in different plant matrices due to the lack of availability of all reference standards. More commonly today, anthocyanins are quantified by spectrophotometric methods, which is non‐specific quantification due to a lack of information on the anthocyanin classes present. In this study, a systematic approach has been proposed to address this challenge, utilizing liquid chromatography‐tandem mass spectrometry using precursor ion scan mode for high‐specificity identification and subsequent quantification of the anthocyanin classes. A method was developed for six anthocyanin classes and it was used to quantify anthocyanins in black carrot extract. The initial scan was conducted for all anthocyanin classes, followed by the quantification of the anthocyanin classes detected using the reference standard specific to that class. This approach has demonstrated that precursor ion scan mass spectrometry can serve as a valuable tool for the rapid identification and specific quantification of anthocyanin classes in plants.

Analysis of pentacyclic triterpenoids and phytosterols in cloudberry (Rubus chamaemorus L.) by LC-MS/MS.

Cloudberry (Rubus chamaemorus L.) is a plant rich in various biologically active compounds. In this work, the composition and concentrations of pentacyclic triterpenoids (PCTs) and phytosterols in various parts of cloudberries were studied using high-performance liquid chromatography-tandem mass spectrometry by multiple reaction monitoring mode (MRM, targeted analysis) and precursor ion scan mode (non-targeted analysis and semi-quantitative determination). Moderately polar fractions of cloudberry leaves, sepals and berries extracts are rich in β-sitosterol glycoside and various triterpenoid acids, among which tormentic acid (∼190-1800 μg/g dw part of cloudberries) and its hydroxyl derivatives (∼3.1-470 μg/g dw part of cloudberries) predominate. Non-polar fractions are characterised by the presence of amyrins (0.76-110 μg/g dw part of cloudberries), β-sitosterol (62-750 µg/g dw part of cloudberries) and β-sitosterol glycoside (∼92-360 μg/g dw sepals).

Suspect Screening and Semi-Quantification of Macrolide Antibiotics in Municipal Wastewater by High-Performance Liquid Chromatography—Precursor Ion Scan Tandem Mass Spectrometry

Macrolides are widely used in medicine and veterinary medicine, and are the leading antibiotics in terms of consumption. The release of macrolides and their metabolites into the environment through municipal wastewater can have an adverse effect on aquatic ecosystems and human health. In the present study, a method for the non-targeted screening and semi-quantitative determination of macrolide antibiotics and their derivatives in wastewater based on a combination of chromatographic separation and tandem mass spectrometric detection in precursor ion scan (PrecIS) mode has been proposed. Product ions with m/z 158 and 174 related to specific desosamine fragments were used as diagnostic ions for the PrecIS detection of the macrolide structures without (14- and 15-membered macrocycles) and with a (16-membered macrocycle) glycosylated desosamine moiety, respectively. The combination of the optimized solid phase extraction procedure and HPLC-MS/MS analysis in PrecIS mode allowed for the suspect screening of macrolides in municipal wastewater with limits of detection in the range of 4–150 ng L−1. The developed approach made it possible to detect and tentatively identify in municipal wastewater 17 compounds belonging to the macrolide class, including azithromycin, clarithromycin, josamycin and 14 metabolites with a total concentration of 1450 ng L−1.

Chain-locked precursor ion scanning based HPLC–MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing ω-3 fatty acyl chains

Lipase-catalyzed transesterification of structured phospholipids (sPLs) is a hot topic, but the structural variation of the fatty acyl chains in intact phospholipids at the molecular level remains unclear to date. The present study explored the detailed characteristics of synthesized phospholipids through high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in precursor ion scan mode. The optimal conditions were in-depth inspected and determined for the reaction system, including phospholipase A1 as catalyst, 15% lipase loading, and 1% water content. The sPLs enriched with EPA/DHA were structurally and quantitatively characterized by focusing on the fragments of m/z 301.6 (eicosapentaenoic acid, EPA) and m/z 327.6 (docosahexaenoic acid, DHA), and the results were statistically analyzed using partial least squares discriminant analysis and clustered heatmap hierarchical clustering analysis. PC 38:6 (18:1/20:5), PC 38:7 (18:2/20:5), PC o-40:6 (o-18:0/22:6), and PE 40:8 (18:2/22:6) etc. were revealed as the main variables that were active in the reaction.

Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers

Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.

Precursor ion approach for simultaneous determination of nonethoxylated and ethoxylated alkylsulfate surfactants

We present a new liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of sodium lauryl sulfate and sodium laureth sulfate homologues in the range of alkyl chain length C12–C16 with 0–5 ethoxy groups. The method is based on scanning the precursor ions fragmenting to m/z 80 and 97 (Precursor Ion Scanning mode), which makes it specific for species with easily cleavable sulfate groups. By monitoring fragmentation of thus discovered quasi-molecular ions we were able to unequivocally identify all sulfate species present in complex mixtures of alkyl and alkyl-ether sulfates with molecular weight ranging from 200 to 600 m/z. Because of the intrinsic sulfate-sensitivity, the presented method can be also applied to non-sodium salts of alkyl- and alkyl-ether sulfates (e.g. ammonium, mono- or triethanolamine, etc.), which are often used by cosmetic manufacturers to justify the misleading SLS- and SLES-free claims (where SLS and SLES refer to sodium lauryl sulfate and sodium laureth sulfate, respectively). The use of reversed phase liquid chromatography (RPLC) column with C4 instead of C18 shortened significantly the overall analysis time and allowed us to use a semiquantitative method (based on single standard for Quantitative Analysis of Multi-component System, QAMS) to determine several SLS and SLES homologues in one run with the limit of quantification (LOQ) = 0.4 µg/mL and of detection (LOD) in the range 0.12–0.97 µg/mL. The method was successfully applied to 17 commercially available cosmetic/household products allowing verification of their manufacturers’ declarations.

Characterization of drug-related impurities using UPLC-QTrap-MS/MS in three scan modes: Application to daptomycin

A new non-targeted screening strategy was developed for the detection and relative quantification of known and potentially unknown drug-related impurities in the active pharmaceutical ingredient daptomycin using ultra-high performance liquid chromatography, coupled with hybrid triple quadrupole-linear ion trap electrospray ionization mass spectrometry in three different modes. The precursor ion scan mode was first applied using the m/z transitions of 159.2, 313.2, and 341.2 for rapid screening of all daptomycin-related impurities. Precursor ions detected at specific retention times were selected, fragmented, and focused in enhanced product ion scan mode to obtain secondary ion mass spectra and identify drug-related impurities. Finally, each potential compound was relatively quantified in multiple reaction monitoring mode. Repeatability experiments suggest that this impurity profiling method is a reliable strategy for identifying unknown impurities in daptomycin and other drugs.

Untargeted Screening of EPA/DHA Structured Phospholipids in Krill Oil by Chain-Lock-Driven Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry.

Eicosapentaenoic and docosahexaenoic acids structured phospholipids (PLEPA/DHA) have multiple biochemical and pharmacological effects on human health. In this study, EPA and DHA chains were locked under precursor ion scan (PreIS) mode for untargeted screening PLEPA/DHA in krill oil using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). The effect of collision energy and declustering potential on the fragmentation of EPA (m/z 301.2) and DHA (m/z 327.2) chains was studied. A total of 33 PLEPA/DHA were characterized (sn-1/sn-2) and quantified using regression models, including 16 PCEPA/DHA, 11 PEEPA/DHA, and 6 PIEPA/DHA. Afterward, this method was validated in terms of linearity (≥0.9978), sensitivity (LOD ≤ 4.02 μg·L-1), precision (RSDintraday ≤ 4.71%), and recovery (≥78.9%). Finally, the performance of HILIC-PreIS-MS/MS was compared with those of conventional methods, and the results indicated its superiority in selective screening PLEPA/DHA in krill oil.

A novel HPLC-MS/MS approach for the identification of biological thiols in vegetables

Thiols are important natural molecules with diverse functions, ranging from acting as antioxidants that prevent chronic diseases to contributing aromas to foods and beverages. Biological thiols such as glutathione are of particular interest due to their functional roles, which include helping maintain cellular redox homeostasis and detoxifying reactive oxygen species. However, knowledge of thiol metabolism in plants is limited to studying known compounds, whereas other important thiol-containing metabolites could also exist. This work aimed to develop a new analytical approach for screening of thiols in plants, using four vegetal examples and beginning with HPLC-MS/MS in precursor ion scan mode, after extraction and thiol-specific derivatisation with 4,4′-dithiodipyridine (DTDP). Compound identity for prospective thiols was then proposed using HPLC with high resolution MS, and verified with authentic standards. This approach could lead to prospecting studies that identify thiols with potential roles in metabolic pathways, nutritional value of vegetables, or flavouring of foods.

Screening and semi-quantitative determination of pentacyclic triterpenoids in plants by liquid chromatography-tandem mass spectrometry in precursor ion scan mode.

Pentacyclic triterpenoids (PCTs) are secondary plant metabolites. They are of exceptional interest as biologically active substances and raw materials for a wide range of medications. Thus, the development of a methodology for rapid screening of PCTs in plant biomass is an important task. The goal of this work was to develop an approach for simultaneous screening and semi-quantitative determination of PCTs in plant tissues by liquid chromatography-tandem mass spectrometry with a precursor ion scan (PrecIS). Pressurised liquid extraction (PLE) with methanol was used for the isolation of PCTs from plant biomass. Screening and semi-quantitative determination of PCTs in the obtained extracts were carried out by reversed phase high-performance liquid chromatography-tandem mass spectrometry in a PrecIS mode. The product ion at m/z 95 with collision energy of 40 V was used as a diagnostic ion to identify PCTs by the PrecIS mode. In plant materials, 26 PCTs and their derivatives, such as PCTs esters and glycosides, were detected and identified. Calculation of the relative response factor for nine available PCTs showed that using a betulin calibration curve allows us to estimate the semi-quantitative content of PCTs and their derivatives in plant PLE extracts. The developed approach can be applied for simultaneous untargeted screening and semi-quantitative determination of PCTs and their derivatives in various plants at sub-parts per million levels.

Determination of picomolar concentrations of thiol compounds in natural waters and biological samples by tandem mass spectrometry with online preconcentration and isotope-labeling derivatization

We present a sensitive, selective and robust method for the determination of 14 thiol compounds in aqueous samples. Thiols were derivatized with ω-bromoacetonylquinolinium bromide (BQB) and its deuterium labeled equivalent D7-ω-bromoacetonylquinolinium bromide (D7). Derivatized thiols were preconcentrated by online solid-phase extraction (SPE) followed by liquid chromatography separation and electrospray ionization tandem mass spectrometry determination (SPE/LC-ESI-MS/MS). The robustness of the method was validated for wide ranges in pH, salinity, and concentrations of sulfide and dissolved organic carbon (DOC) to cover contrasting natural water types. The limits of detection (LODs) for the thiols were 3.1–66 pM. Between 6 and 14 of the thiols were detected in different natural sample types at variable concentrations: boreal wetland porewater (0.7–51 nM), estuarine sediment porewater (50 pM–11 nM), coastal sea water (60 pM–16 nM), and sulfate reducing bacterium cultures (80 pM–4 nM). MS/MS fragmentation of the compounds produces two pairs of common product ions, m/z 130.2/137.1 and 218.1/225.1, which enables scanning for unknown thiols in precursor ion scan mode. Using this approach, we identified cysteine, mercaptoacetic acid, N-acetyl-L-cysteine and sulfurothioic S-acid in boreal wetland porewater. The performance of the developed method sets a new state of the art for the determination of thiol compounds in environmental and biological samples.

Tandem Analysis by a Dual-Trap Miniature Mass Spectrometer.

Miniature mass spectrometers are of an increasing interest for in-situ analyses, and their coupling with the ambient ionization sources is a valid path for direct analysis of complex samples. In this study, a miniature mass spectrometer using discontinuous atmospheric pressure interface was developed with a dual-LIT (linear ion trap) configuration. The comprehensive scan modes were enabled for tandem mass spectrometry analysis, which are critical for high-quality qualitative and quantitative analysis. A real-time pressure control was implemented to facilitate the ion transfer and collision induced dissociation (CID). Beam-type CID could be performed for tandem analysis at a high number of stages. In-trap CID at high q could also be performed with the fragment ions accumulated in a second trap. A precursor ion scan mode for analyzing target analytes has also been demonstrated.

Targeted profiling and relative quantification of benzoyl diterpene alkaloids in Aconitum roots by using LC-MS/MS with precursor ion scan.

Benzoyl aconite alkaloids have myocardial protective effects at a low dose and produce toxic effects at high dose. Due to lack of enough reference compounds, most of the benzoyl alkaloids had few concerns, except the typical ones, i.e. aconitine, mesaconitine, and hypaconitine. To rapidly screen out and quantify benzoyl alkaloids, a high performance liquid chromatography combined with tandem mass spectrometry was proposed based on precursor ion scanning mode. First, a diagnostic ion at m/z 105 corresponding to benzoyl group was observed by using tandem mass spectrometry, which could be used for the rapid identification of benzoyl alkaloids. The targeted screening of these alkaloids was then conducted by using precursor ion scan of characteristic ion at m/z 105. Shengfuzi (the lateral root of A. carmichaelii) was taken as example, and 24 benzoyl-containing alkaloids were identified. The six major alkaloids including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine were determined in the precursor ion scan mode by the standard curve method. Reliable linearity, sensitivity, precision, accuracy, and repeatability were obtained and validated. Then the relative response factors between these six analytes were calculated, which were not more than two times using any alkaloid as reference. Thus, the other 18 alkaloids lacking reference compounds were relatively quantified. This approach provides a useful tool for rapid identification and quantitative analysis of toxic benzoyl alkaloids, and also an efficient method for the safety assessment of Aconitum roots.

Development of quantitation method for glycated aminophospholipids at the molecular species level in powdered milk and powdered buttermilk

The Maillard reaction is a nonenzymatic glycation reaction between a reducing sugar and a free amino group, known to naturally occur during heat processing of food. In this study, we especially focused on phosphatidylethanolamine (PE)-linked Amadori products (Amadori-PE) in powdered milk, since the analysis of these products at the molecular species level has not yet been evaluated. Analysis of Amadori-PE was conducted by using liquid chromatography-tandem mass spectrometry in three different modes. The main Amadori-PE species in a powdered milk sample were first identified as 34:1, 36:1, 36:2 and 36:3 in the total ion current mode. Additionally, by using the characteristic product ions observed in the presence of sodium, we quantified the main Amadori-PE species in the multiple reaction monitoring mode, and evaluated their total concentrations in the precursor ion scan (PIS) mode for the first time. Powdered milk contained much Amadori-PE with concentrations ranging from 4.3 to 8239 mg/100 g, quantified by the PIS mode. The newly developed methods represent powerful tools for detailed analysis of glycated lipids including Amadori-PE in powdered milk, which may further be applied to research relating to infant food and nutrition.

Implementation of Precursor and Neutral Loss Scans on a Miniature Ion Trap Mass Spectrometer and Performance Comparison to a Benchtop Linear Ion Trap.

Implementation of orthogonal double resonance precursor and neutral loss scans on the Mini 12 miniature rectilinear ion trap mass spectrometer is described, and performance is compared to that of a commercial Thermo linear trap quadropole (LTQ) linear ion trap. The ac frequency scan version of the technique at constant rf voltage is used here because it is operationally much simpler to implement. Remarkably, the Mini 12 shows up to two orders of magnitude higher sensitivity compared to that of the LTQ. Resolution on the LTQ is better than unit at scan speeds of ~ 400Th/s, whereas peak widths on the Mini 12, on average, range from 0.5 to 2.0Th full width at half maximum and depend heavily on the precursor ion Mathieu q parameter as well as the pump down time that precedes the mass scan. Both sensitivity and resolution are maximized under higher pressure conditions (short pump down time) on the Mini 12. The effective mass range of the product ion ejection waveform was found to be 5.8Th on the Mini 12 in the precursor ion scan mode vs. that of 3.9Th on the LTQ. In the neutral loss scan mode, the product ion selectivity was between 8 and 11Th on the Mini 12 and between 7 and 8Th on the LTQ. The effects of nonlinear resonance lines on the Mini 12 were also explored. Graphical Abstract ᅟ.

Precursor Ion-Scan Mode-Based Screening Strategy for New Amorfrutin Derivatives from Amorpha fruticosa by UPLC–QqQ–MS and UPLC–Q-TOF–MS

This study presents a screening strategy for unknown compounds by coupling UPLC–QqQ–MS and UPLC–Q-TOF–MS analyses. Amorfrutins, potential PPARγ agonists in Amorpha fruticosa, were selected as the research target. First, MS/MS analysis of three amorfrutin references (AA, AB, and AC) by Q-TOF revealed the mass-spectrometric fragmentation patterns of amorfrutins, and m/z 225 was recognized as a characteristic and common fragment ion of amorfrutins. Then, unknown amorfrutins were screened in the fruits of A. fruticosa by UPLC–QqQ–MS operating in precursor ion-scan mode, and the product ion was set as m/z 225 for screening potential amorfrutins. According to the high-resolution MS–MS spectral data provided by UPLC–Q-TOF–MS, the molecular formula of potential amorfrutins and their respective characteristic fragments were calculated. Finally, three unknown amorfrutins, namely, amorfrutin D and two new amorfrutins, were identified. As far as we know, this is the first study to rapidly identify unknown compounds by combined application of QqQ–MS precursor ion scan and Q-TOF–MS formula calculation. This research strategy will also be effective for the discovery of other trace natural products and metabolites.

Rapid discovery of cyclopamine analogs from Fritillaria and Veratrum plants using LC-Q-TOF-MS and LC-QqQ-MS

Cyclopamine, an inhibitor of the Hedgehog (Hh) signaling pathway, has been paid much attention in treating a wide variety of tumors. However, isolation and purification of cyclopamine analogs from medicinal plants remain challengeable. We herein proposed an efficient strategy using liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) and liquid chromatography triple-quadrupole mass spectrometry (LC-QqQ-MS) for rapid screening and targeted isolation of cyclopamine analogs in Fritillaria and Veratrum plants. Firstly, fifteen reference compounds were characterized by LC-Q-TOF-MS and their characteristic fragment ions were summarized. Secondly, according to the characteristic fragment ions at m/z 67.1, 84.1, 109.1 and 114.1, rapid chemical screening of plant extracts was carried out by LC-QqQ-MS using precursor ion scan mode and 69 pre-target compounds were screened out. Thirdly, 24 real target compounds were verified by LC-Q-TOF-MS based on relative abundances (over 20%) of characteristic fragment ions. Fourthly, the targeted isolation of Fritillaria ussuriensis bulb and Veratrum dahuricum rhizome afforded a novel cyclopamine analog namely 15β-hydroxy-23-isopengbeisine B as well as four known ones, whose structures were determined by nuclear magnetic resonance (NMR) analysis. Additionally, these five analogs were evaluated for the inhibitory activity of Hh signaling pathway in NIH/3T3 cell and cytotoxicity in PANC-1 and HepG2 cells. These results indicated that the proposed strategy was reliable for rapid discovery and targeted isolation of important natural products from chemically complex plant matrices.

Precursor Ion Scan Mode-Based Strategy for Fast Screening of Polyether Ionophores by Copper-Induced Gas-Phase Radical Fragmentation Reactions

The potential of copper(II) to induce gas-phase fragmentation reactions in macrotetrolides, a class of polyether ionophores produced by Streptomyces species, was investigated by accurate-mass electrospray tandem mass spectrometry (ESI-MS/MS). Copper(II)/copper(I) transition directly induced production of diagnostic acylium ions with m/z 199, 185, 181, and 167 from α-cleavages of [macrotetrolides + Cu]2+. A UPLC-ESI-MS/MS methodology based on the precursor ion scan of these acylium ions was developed and successfully used to identify isodinactin (1), trinactin (2), and tetranactin (3) in a crude extract of Streptomyces sp. AMC 23 in the precursor ion scan mode. In addition, copper(II) was also used to induce radical fragmentation reactions in the carboxylic acid polyether ionophore nigericin. The resulting product ions with m/z 755 and 585 helped to identify nigericin in a crude extract of Streptomyces sp. Eucal-26 by means of precursor ion scan experiments, demonstrating that copper-induced fragmentation reactions can potentially identify different classes of polyether ionophores rapidly and selectively.

Insights on profiling of phorbol, deoxyphorbol, ingenol and jatrophane diterpene esters by high performance liquid chromatography coupled to multiple stage mass spectrometry

This paper reports our effort to develop a comprehensive HPLC-MSn-based dereplication strategy for phorbol ester (PE), deoxyphorbol ester (dPE) and ingenol ester (IE) profiling in plant extracts. This strategy is composed of two sequential analysis exploiting specific hybrid triple quadrupole/linear ion trap instrument modes. A first run was performed using a multiple reaction monitoring (MRM) mode targeting fragmentation of PE and dPE/IE coupled with the acquisition of MS2 spectrum for the ions at m/z 311 and m/z 313, respectively. A second run was then completed based on precursor ion scan mode (PIS) and automatic MS2 acquisition for each quasimolecular ion. The developed approach was used to investigate ten Euphorbia extracts showing bioactivity against chikungunya virus replication. Experiments allowed partial annotation of three dPE/IE but no PE was detected. Results suggested that other types of diterpene esters displayed PE- and dPE/IE-like fragmentations. The study of jatrophane ester (JE) standards by CID fragmentation using low and high resolution mass spectrometry confirmed this hypothesis, highlighting challenges and difficulties of diterpene esters profiling within plant extracts. Nonetheless, the present LC–MSn method can be easily adapted to profile other types of diterpene esters.

Development of a mass-spectrometry-based lipidomics platform for the profiling of phospholipids and sphingolipids in brain tissues.

This article describes the development of a lipidomic platform consisting of a 4000 QTRAP mass spectrometer and a self-installed sample inlet system to indentify and quantify 12 phospholipid and five sphingolipid classes from lipid-rich brain tissues of mouse, duck, and salmon. The total mass spectrometry analysis time per sample was 30min, including 14min for direct infusion for phospholipids and sulfatide in precursor ion scanning mode or neutral loss scanning mode, and 16min for liquid-chromatographic separation of ceramide, sphingomyelin, monohexosylceramide, and dihexosylceramide in multiple reaction monitoring mode. The method was fully validated in terms of linearity, detectability, recovery, and repeatability, with satisfactory results for all targets. We individually quantified 307, 308, and 330 lipid species from 17 lipid subclasses, and obtained total amounts of 57.2, 61.7, and 53.1mg/g wet brain for mouse, duck, and salmon tissues, respectively. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were the major lipids in all the brain samples, whereas phosphatidylinositol occurred at a relatively higher level in the salmon sample. For phospholipids, sphingolipids, and minor lysophospholipids, differences in the identity of the molecular species, their distributions, and their relative amounts as well as the contribution of each lipid subclass to the whole polar lipidome were found. Palmitic acid (16:0), stearic acid (18:0), lignoceric acid (24:0), oleic acid (18:1), nervonic acid (24:1), arachidonic acid (20:4), and docosahexaenoic acid (22:6) were found as the main saturated, monounsaturated, and polyunsaturated fatty acids in samples from the different species, but eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) were more abundant in the salmon brain sample. The results are in good agreement with those in previous reports obtained from the relevant tissues, providing a reliable basis that could be extended to clinical research and resource evaluation. Graphical Abstract Methodology for phospholipids and sphingolipids profiling in brain tissues.